--- The chief goal is to understand how the hormone vasopressin regulates water excretion by the kidney. Vasopressin's action is mediated through regulation of the molecular water channel aquaporin-2. Based on our studies a decade ago, it is now clear that vasopressin regulates aquaporin-2 in a time frame of seconds to minutes by altering the distribution of the water channel aquaporin-2 between the plasma membrane and the cytoplasm via vesicular trafficking. Trafficking of aquaporin-2 to the plasma membrane renders the cells permeable to water. We are presently using a systems approach to address the mechanisms involved. For this approach, we are integrating protein mass spectrometry, DNA microarrays, deep sequencing, mathematical modeling and physiological methods. This work has produced 10 publications over the last 12 months. --- There are two major areas of focus currently: 1) elucidation of the signaling network for vasopressin responses in the renal collecting duct;and 2) understanding how vasopressin regulates the abundance of the aquaporin-2 water channel and other proteins in the renal collecting duct. --- In the first area, we have already published a series of papers showing phosphoproteomic responses to vasopressin in the inner medullary collecting duct of rat, in the thick ascending limb of rat, and in cultured cortical collecting duct cells (see reference list). We have completed a dynamic study of the phosphoproteomic response to vasopressin using iTRAQ to track changes in thousands of individual phorphorylation sites over a 15 minute time period after vasopressin exposure (in review). We have also completed work on new methodology for profiling individual protein kinases with regard to the sequence preferences in the target substrate proteins (in review). The current thrust in the first area of focus is to map individual protein kinases to regulated phosphorylation targets in the collecting duct. --- In the second area of focus, we have published an article describing studies in which we have carried out global profiling of vasopressin-induced changes in both protein abundance and transcript abundance in the same collecting duct cells (Khositseth et al. see reference below). In general, there was a low correlation between these two measures for most proteins. In particular, a large numbers of proteins exhibited changes in protein abundance in response to vasopressin without corresponding changes n transcript abundances. The implication is that there is regulation of translation or protein stability for a large number of proteins. To follow this up, we have carried out global profiling of protein half lives using dynamic SILAC (in preparation for publication) revealing that only a small fraction of post-translationally regulated proteins are regulated by altering half life including aquaporin-2. We are now using SILAC to determine translation rates for all proteins across the proteome. An additional focus is on transcriptional regulation. We have carried out global profiling of vasopressin-induced nuclear translocation (under review), identifying a relatively small number of transcription factors that move into the nucleus in response to vasopressin. Also, we are finishing up a study to identify nuclear proteins that become phosphorylated in response to vasopressin to identify additional candidate proteins that may play roles in vasopressin-regulated transcription in collecting duct cells (in preparation for publication). Recent Papers: 1: Zhao B, Knepper MA, Chou CL, Pisitkun T. Large-Scale Phosphotyrosine Proteomic Profiling of Rat Renal Collecting Duct Epithelium Reveals Predominance of Proteins Involved in Cell Polarity Determination. Am J Physiol Cell Physiol. 2011 Sep 21. Epub ahead of print PubMed PMID: 21940666. 2: van Balkom BW, Pisitkun T, Verhaar MC, Knepper MA. Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases. Kidney Int. 2011 Aug 31. doi: 10.1038/ki.2011.292. Epub ahead of print PubMed PMID: 21881557. 3: Stdkilde L, Nrregaard R, Fenton RA, Wang G, Knepper MA, Frkir J. Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2. Am J Physiol Renal Physiol. 2011 Jul;301(1):F226-35. Epub 2011 Apr 27. PubMed PMID: 21525134;PubMed Central PMCID: PMC3129890. 4: Hoffert JD, Pisitkun T, Knepper MA. Phosphoproteomics of vasopressin signaling in the kidney. Expert Rev Proteomics. 2011 Apr;8(2):157-63. Review. PubMed PMID: 21501009. 5: Feric M, Zhao B, Hoffert JD, Pisitkun T, Knepper MA. Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule. Am J Physiol Cell Physiol. 2011 Apr;300(4):C755-70. Epub 2011 Jan 5. PubMed PMID: 21209370;PubMed Central PMCID: PMC3074622. 6: Khositseth S, Pisitkun T, Slentz DH, Wang G, Hoffert JD, Knepper MA, Yu MJ. Quantitative protein and mRNA profiling shows selective post-transcriptional control of protein expression by vasopressin in kidney cells. Mol Cell Proteomics. 2011 Jan;10(1):M110.004036. Epub 2010 Oct 12. PubMed PMID: 20940332; PubMed Central PMCID: PMC3013460. 7: Gunaratne R, Braucht DW, Rinschen MM, Chou CL, Hoffert JD, Pisitkun T, Knepper MA. Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15653-8. Epub 2010 Aug 16. PubMed PMID: 20713729; PubMed Central PMCID: PMC2932563. 8: Pisitkun T, Hoffert JD, Saeed F, and Knepper MA. NHLBI-AbDesigner: An online tool for design of peptide-directed antibodies. Am J Physiol Cell Physiol. 2011 Sep 28. Epub ahead of print 9: Hwang S, Gunaratne R, Rinschen MM, Yu MJ, Pisitkun T, Hoffert JD, Fenton RA, Knepper MA, Chou CL. Vasopressin increases phosphorylation of Ser84 and Ser486 in Slc14a2 collecting duct urea transporters. Am J Physiol Renal Physiol. 2010 Sep;299(3):F559-67. Epub 2010 Jun 24. PubMed PMID: 20576681;PubMed Central PMCID: PMC2944290. 10: Gonzales PA, Zhou H, Pisitkun T, Wang NS, Star RA, Knepper MA, Yuen PS. Isolation and purification of exosomes in urine. Methods Mol Biol. 2010;641:89-99. PubMed PMID: 20407943. 11: Da Silva N, Pisitkun T, Belleanne C, Miller LR, Nelson R, Knepper MA, Brown D, Breton S. Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting. Am J Physiol Cell Physiol. 2010 Jun;298(6):C1326-42. Epub 2010 Feb 24. PubMed PMID: 20181927;PubMed Central PMCID: PMC2889637. 12: Rinschen MM, Yu MJ, Wang G, Boja ES, Hoffert JD, Pisitkun T, Knepper MA. Quantitative phosphoproteomic analysis reveals vasopressin V2-receptor-dependent signaling pathways in renal collecting duct cells. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3882-7. Epub 2010 Feb 5. PubMed PMID: 20139300;PubMed Central PMCID: PMC2840509. 13: Fernndez-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA. Tamm-Horsfall protein and urinary exosome isolation. Kidney Int. 2010 Apr;77(8):736-42. Epub 2010 Feb 3. PubMed PMID: 20130532. 14: Xie L, Hoffert JD, Chou CL, Yu MJ, Pisitkun T, Knepper MA, Fenton RA. Quantitative analysis of aquaporin-2 phosphorylation. Am J Physiol Renal Physiol. 2010 Apr;298(4):F1018-23. Epub 2010 Jan 20. PubMed PMID: 20089674;PubMed Central PMCID: PMC2853310. 15: Bansal AD, Hoffert JD, Pisitkun T, Hwang S, Chou CL, Boja ES, Wang G, Knepper MA. Phosphoproteomic profiling reveals vasopressin-regulated phosphorylation sites in collecting duct. J Am Soc Nephrol. 2010 Feb;21(2):303-15. Epub 2010 Jan 14. PubMed PMID: 20075062;PubMed Central PMCID: PMC283